Characterization of the rat pituitary capsule: Evidence that the cerebrospinal fluid filled the pituitary cleft and the inner side of the capsule

In humans, the pituitary gland is covered by a fibrous capsule and is considered a continuation of the meningeal sheath. However, in rodents some studies concluded that only the pars tuberalis (PT) and pars nervosa (PN) are enwrapped by the pia mater, while others showed that the whole gland is covered by this sheath. At PT the median eminence subarachnoid drains cerebrospinal fluid (CSF) to its cisternal system representing a pathway to the hypothalamus. In the present study we examined the rat pituitary capsule to elucidate its configuration, its physical interaction with the pituitary border and its relationship with the CSF. Furthermore, we also revisited the histology of the pituitary cleft and looked whether CSF drained in it. To answer such questions, we used scanning and transmission electron microscopy, intracerebroventricular infusion of Evan´s blue, fluorescent beads, and sodium fluorescein. The latter was measured in the pars distalis (PD) and various intracranial tissues. We found a pituitary capsule resembling leptomeninges, thick at the dorsal side of the pars intermedia (PI) and PD, thicker at the level of PI in contiguity with the PN and thinner at the rostro-ventral side as a thin membrane of fibroblast-like cells embedded in a fibrous layer. The capsule has abundant capillaries on all sides. Our results showed that the CSFs bathe between the capsule and the surface of the whole gland, and ciliate cells are present in the pituitary border. Our data suggest that the pituitary gland intercommunicates with the central nervous system (CNS) through the CSF.

Introduction this report were approved by the Cinvestav Animal Used and Care Ethical Committee (CIC-UAL, # 0267-05), following the Mexican Official Rule NOM-062-ZOO-1999 and the Guide from the National Institutes of Health (NIH-USA, #8023). There were 35 rats used in the present study.

Pituitary glands
The pituitary glands were obtained from perfused animals or from decapitated animals. The perfusion was developed in rats anaesthetized with 60 mg/kg body weight sodium pentobarbital (Pentobarbital1, Aranda, MX) and perfused through the left heart ventricle with 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer (pH 7.4) or 4% paraformaldehyde plus 3% sucrose in phosphate-buffer saline (PBS) 100 ml (pH 7.4). The large vessels, both vena cava and aorta descendent, were clamped prior to perfusion. In some experiments, PBS was perfused first followed by the fixer solution. The rate of perfusion was 1 drop/sec of 100 ml. After fixation, the pituitary gland was dissected, immersed in fixer solution, and processed for microscopic observation. Some glands were obtained from decapitated rats and the glands were fixed by immersion with 2.5% glutaraldehyde in 0.15 M sodium cacodylate buffer (pH 7.4) for 4 h at 4˚C.

Scanning Electron Microscopy
Pituitary glands obtained after the rats were perfused with glutaraldehyde were separated into two groups: a group of glands transversally cut and the other used the intact gland, the latter to observe the whole surface of the gland. Both groups of glands were immersed in the same fixative plus sucrose 2% at 4˚C overnight, followed by a treatment with 1% tannic acid in the same fixative solution at room temperature for 2 h in darkness. Then, the glands were rinsed and postfixed with 2% OsO 4 plus 2% sucrose in cacodylate buffer for 2 h followed by an incubation with 1% OsO 4 overnight at 4˚C. After several washes the glands or the cuts were dehydrated with ethanol (5 to 100%) and subsequently infiltrated with isoamyl acetate (Sigma, Burlington, MA, USA) and dried with hexamethyldisilazane (HMDS, Electron Microscopy Sciences, Philadelphia, PA, USA) in a desiccator for 48 h. The samples were gold-sputtered and analysed under a scanning electron microscope (JSM-6510LV, JEOL, Tokyo, Japan). In some experiments were used the Tanaka´s technique, using a double-sided adhesive carbon tape the tissue is attached to it and then its pull. This technique was used to observe what was under the fibrous layer covering the PD.

Transmission Electron Microscopy
Pituitary glands obtained from decapitated rats were cut into small pieces of approximately 1-2 mm, placed in glutaraldehyde solution and postfixed with 1% OsO 4 in cacodylate buffer 0.15 M 2 h, at room temperature. Then, the tissues were washed and dehydrated in a graded series of ethanol from 30% to 100%, followed by propylene oxide and embedded in Embed 812 (Electron Microscopy Sciences). Ultrathin sections (60-90 nm) were mounted on nickel grids (Polysciences Inc, Philadelphia, PA, USA). The sections were rehydrated in distilled water drops for 10 min and counterstained with 4% uranyl acetate for 20 min, followed by 2.5% lead citrate for 7 min, washed several times with distilled water and dried. Sections were examined, and electron micrographs were taken by using an electron microscope (Crossbeam 550, Gemini 2, Zeiss, Jena, Germany). Semithin sections (0.5 μm) were also obtained, stained with toluidine blue and mounted in Entellan (Merck Millipore, Darmstadt, Germany).

Intracerebral ventricular injection
Anaesthetized rats were held in a stereotaxic frame (Stoelting Co, Chicago, IL, USA) to trepan their skulls with a dental drill at the appropriate location. A 0.5% Evan´s blue solution in PBS (Sigma-Aldrich, St. Louis, MO, USA) or 6 mg/ml sodium fluorescein (NaFluo) in PBS was injected into the left lateral ventricle at the following coordinates: AP, 58.2 mm from the interaural midpoint; ML, 20.3 mm from the intraparietal suture; DV, 17 mm from dura mater. The perfusion rate was 0.2 μl/min for 10 min using an injection microperfusion pump (Mod. 100, Stoelting Co, USA). After Evan´s blue or NaFluo perfusion the needle was rested for 7 min and then withdrawn in steps of 1 mm/min [14]. In some experiments, polystyrene pearls of 2or 0.2-μm diameter were perfused using the same protocol.

Light and fluorescence microscopy
After the intracerebroventricular infusion of Evans blue, NaFluo or polystyrene pearls the rats were perfused intracardially with PBS followed by paraformaldehyde 4% and sucrose 3% in PBS. In some experiments, 220 μl of Evan´s blue solution or 1.0% toluidine blue solution were administered through the left heart ventricle at the end of the fixation solution, followed by the same procedure used for the dyes and pearls administered intracerebroventricularly [15]. The dura mater sack that encapsulates the pituitary was removed in situ, the gland was washed 3 times with 1 ml PBS and then continued with the histological processes. The dissected pituitary was placed in 20% sucrose in PBS for 8 h at 4˚C, embedded in a cryoprotect gel (Freeze Mount, BBC Biochemical, Mount Vernon, WA, USA) and cut into 50-μm thickness slides using a cryostat (Leica, Germany). The tissue slides were observed, and the images were acquired with a Leica TCS SP8 424 laser confocal microscope, equipped with a Leica HCX PL FLUOTAR 10x/0.30 DRY and a Leica HC PL APO CS2 63x/1.40 oil objective (Leica, Jena, Germany). Fluorescence microscopy images of the 2 and 0.2 μm polystyrene pearls were obtained with an Axio Observer Z1 microscope equipped with a Zeiss ACHROPLAN 100x/ 1.25Oil PH3 objective and filters: 38H (excitation 470/40, emission 525/50) and 49 (excitation 365, emission 445/50) (Zeiss, Jena, Germany). Images were obtained with steps of 0.5 μm and then processed using the Image J free software v1.47 and the plugging 3D (NIH; USA). For the light microscopy observations, rats were perfused with paraformaldehyde 4% plus sucrose 3% in PBS, the brain and pituitary dissected and processed for paraffin embedded-HE stain standard technique. The sections were observed and photographed using an Eclipse 80i microscope and NIS-Elements software (Nikon, Tokyo, Japan).

Tissue sodium fluorescein measurement
The tissue content of NaFluo was quantified according to Schoch et al. [16] with some modifications. Briefly, the pituitary gland without PI and PN, optic nerves, cerebellar lobule IV-V cortex, and media eminence were obtained after NaFluo intracerebroventricular infusion, placed in 200 μl PBS at pH 7.4 and immediately frozen. Then, the tissues were ground in liquid N 2 , 1.2 ml absolute ethanol was added for protein precipitation, and the samples were centrifuged at 13,000 rpm for 20 min at 4˚C. The NaFluo concentration was measured in the supernatant at 520 nm emission with an excitation wavelength of 485 nm in a Spectrofluorometer (Infinite 200Pro, Tecan, Männendorf, Switzerland). The protein precipitate was determined by the Lowry method (BioRad, Watfor, UK). Data are presented as fluorescein arbitrary units per protein mg. After intracerebroventricular infusion of NaFluo the tissues were photographed using a smartphone attached to the eyepiece ocular 10X of a stereomicroscope (SMZ645, Nikon, Tokyo, Japan).

Statistical analysis
Values of tissue fluorescence intensity are expressed as the mean ± standard error of the mean (S.E.M). Differences between the tissue endogenous fluorescence and the NaFluo in the tissues were analysed by one-way analysis of variance (ANOVA) followed by Holm-Sidak´s multiple comparisons test (GraphPad Prism 7.00, San Diego, CA, USA).  The pituitary has a gross external envelope or capsule that covers part of the PN and the PI and extends to the PD (Fig 2A). Fig 2B and 2C shows that the envelope is invaginated at the borders between the PN and PI. In addition, on the PI side, there are many blood vessels. A transversal cut at the middle of the PN exhibits a space between both pituitary lobules where the external envelope comes in (Fig 2D). If one moves towards the PD where the external envelope gets thinner and makes a zoom in this area (boxed area), multiple vessels could be observed (Fig 2E and 2F).  Fig 2B and shows details of the thicker folded external envelope with fibrous borders and a complex surface, as can be observed in Fig 3B. Fig 3C shows a semithin section of the PI and its capsule whose structure resembles leptomeninges. At the border of the PI, a thin layer with fibroblast-like cells separates the glandular tissue from the capsule. It is possible to observe capillary vessels surrounded by pericytes and fibroblast-like cells embedded in loosely arranged connective tissue where cells with a less differentiated morphology are present (Fig 3C). At the external border, the capsule appeared denser and thicker with many erythrocytes among the connective tissue, suggesting a very thin wall of capillaries at this level (Fig 3C and 3D). Additionally, there were long and slender cells

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The rat pituitary capsule

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The rat pituitary capsule Semithin sections of the PI covered by the capsule, which is similarly to lose connective tissue. The PI consists of a relatively large amount of amorphous extracellular matrix with capillaries and different types of cells. Moreover, the PI and the capsule are separated by a thin fibrous layer and fibroblast-like cells from the capsule, whose external limit looks denser and thicker, with fusiform cells and peripheral capillaries. Some erythrocytes were observed on the capsule surface. The rectangle is observed as a higher magnification of the capsule in (d). (e) Transmission electron microscopy (TEM) of the loose connective tissue with fibrocytes forming well-defined and clear spaces where macrophages-like cells with cytoplasmic protrusions were frequently observed. Other less differentiated cells with large nuclei and abundant euchromatin were located outside the spaces. (f) TEM of the external limit of the capsule showing lamellar organization of overlapping cytoplasmic prolongations from fibrocytes. Long and flattened fibroblast-like cells with elongated nuclei, such as fibroblast cells, at the innermost aspect of the capsule (Fig 3C). In summary, the semithin section photomicrographs allowed us to distinguish various types of cells according to their size, nucleus shape, and chromatin arrangement (Fig 3C and 3D). Fig  3E shows details at the transmission electron microscopy (TEM) of the cells observed in the loosely arranged connective membrane where at least three different types of cells can be identified: cells with thin membrane protrusions and dentate nucleus and marginated heterochromatin, a macrophage cell; a cell with a larger and paler nucleus than the other cells, a trabecular cell; a fibroblast-like cell with an elongated nucleus surrounded fibrous fibres. The envelope or capsule of the pituitary has an external fibrous layer and fibroblast-like cells densely packed in a fibrous membrane (Fig 3C-3F). Below this fibrous layer, there is the typically complex arachnoid membrane, with intermingled membrane processes rich in mitochondria ( Fig 3G and 3H). Details of the fibroblast-like cells at the outermost border of the capsule are shown in Fig 3I. The PD, at the most posterior level, was also seen in the SEM and was covered by a thick multilayer capsule, constituted of multiple fibrous layers with capillaries, and fibroblast-like cells (Fig 4A-4C). When the capsule is broken using the Tanaka´s technique (Fig 4D), it is possible to observe the thickness of the capsule and the surface of the glandular tissue (Fig 4E and  4F). It is interesting that some cells display a short primary cilium-like structure protruding to the space beneath the capsule (Fig 4G and 4H). However, the capsule is not thicker all around the PD, as could be observed in Fig 2 and in the semithin sections in Fig 4I and 4J. A layer of fibroblast-like cells, with long and slender cytoplasmic processes, was in close apposition with glandular cells, as could be observed at the PI capsule (Fig 4K-4M).
An SEM view of the PD at the more ventral and lateral side shows a sinuous surface of the pituitary with lengthen cords, defined by furrows at the outer edges suggesting the external layer invaginates between the tissue cords ( Fig 5A). An SEM view of the PD at the more ventral and lateral side shows a sinuous surface of the pituitary with lengthen cords of glandular tissue ( Fig 5A). A semithin section shows that the PD cells are arranged mainly in cords between which are large-bore capillaries. At the outermost surface, there is a thin fibrous mesh, like fine reticular fibres with fibroblast-like cells (Fig 5B and 5C). It was also observed that the fibrous membrane protruded to the external space and that it is attached to the superficial tissue (S1 Fig). In addition, peripheral capillaries are frequently observed in this multilayer capsule, and invaginations or septa formation that fuses with the capillary wall are common (Fig 5C). The TEM of a lateral view showed a capsule formed by an extracellular matrix of fibrils and floccular components and cytoplasmic processes of fibroblast-like cells. Additionally, a thin continuous amorphous sheet closely following the basal contour of endocrine cells was identified. This sheet was formed by a lamina densa separated from the cell surface of granular and agranular cells by the lamina lucida or lamina rara (Fig 5D-5G). The membrane at this level viewed by TEM ( Fig 5D) showed a fibrous membrane with floccular material and some elongated cellular processes, where granular cells were separated by a basal membrane, as clearly shown in Fig 5E. In Fig 5E-5G, a space between the PD superficial cells and the capsule is observed, and in Fig 5F and 5G, it is clearly shown that the membrane processes of the capsule cells did not contact the glandular tissue, neither granular cells nor agranular cells.

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The rat pituitary capsule

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The rat pituitary capsule

The epithelial lining of Rathke´s cleft
The PI-side epithelial lining of Rathke´s cleft that covers the PI is formed by a single layer of columnar epithelia in which multiciliate cells predominate (Fig 6A and 6B). However, at the most rostral zone of the PI the epithelial lining changes its cell population. The surface exhibited fewer cilia, and some protuberant apexes with microvilli stood out among ciliated cells (Fig 6C). Areas with a nonciliated or microvilli surface were observed. This zone of the cleft is apparently constituted of epithelial cells whose apical surface carries microfolds, and some present a single or primary cilium (Fig 6C and 6D). Semithin tissue sections showed that the PI-side epithelial lining of the Rathke´s cleft is composed of columnar ciliated and nonciliated cells (with microvilli) and cuboidal cells creating a pseudostratified appearance (Fig 6E and  6F). The epithelium rests on an underlying scarce stroma with capillaries, pericytes and fibroblast-like cells. The epithelium and the melanotrope cells are separated by this stroma or lamina propria from which loose connective tissue septa may extend into the interior of PI, dividing it into small lobules or endocrine cell nests (Fig 6E and 6F). TEM corroborates that the posterior epithelia lining the cleft are columnar pseudostratified (Fig 6G). A basal mitochondrial-rich cell type, which apparently does not reach the lumen or epithelial surface, was covered by the electron-dense apical regions of ciliated cells and cells with microvilli ( Fig 6G). These cells have round, clear, vacuole-like cytoplasmic spaces or macropinocytic vesicles. Near the apical surface, the lateral cell membranes of these two epithelial cell types form typical epithelial junctional complexes (Fig 6H and 6I).
The surface of the PD-side epithelial lining of Rathke´s cleft is characterized by the presence of small, medium, and larger protuberant apexes (Fig 7A-7C), many with microfolds, some with a single or primary cilium (Fig 7C), and few with cilia ( Fig 7B). These apexes are interposed among more flattened and smoother surfaces. Semithin tissue sections ( Fig 7D) and TEM (Fig 7E and 7F) showed that apexes of the luminal extreme of a lining simple epithelium of pseudostratified appearance by alignment at different levels of nuclei. Three types of cells are recognizable: two types of columnar cells, ciliated and with microvilli, whose nuclei are arranged in rows parallel to each other, and one small, superficial cuboidal to flattened cell, with rounded nuclei. Ciliated cells are readily identifiable by their electron-dense bodies, although they also have numerous irregular microvilli between the cilia (Fig 7E and insert). In addition, cells with only microvilli of irregular size and shape are clearly distinguishable from flattened and ciliated cells (Fig 7E and 7F). Typical epithelial junctional complexes were observed among epithelial cells. Interestingly, ciliated cells have abundant mitochondria, cells with microvilli have abundant ribosomes and some have large droplets of colloidal material (Fig 6E).

The CSF, PI and PD
The red fluorescent emission of Evans blue dye was observed at the periphery of the rat pituitary, around PN and PD 10 minutes after its administration through the left heart ventricle (Fig 8A and 8B). It was observed at the periphery of all around the pituitary gland, PN and PD (Fig 8A and 8B) where continuous non-fenestrated capillaries were observed (S2a and S2b  Fig). No red fluorescence was noticed in the endocrine and nervous components of the hypophysis, because Evans blue dye was washed with the procedure used to cryo-protect the gland due to the presence of fenestrated capillaries in the pituitary (S2c and S2d Fig); when this procedure was not used, the red fluorescent dye was observed inside the pituitary (S2e Fig). However, 20 min after finishing the intracerebroventricular administration of Evans blue dye, it was observed inside the pituitary cleft, suggesting that the CSF penetrates this space (Fig 8C and 8D). Moreover, Evans blue remained around the whole pituitary, even after the

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The rat pituitary capsule incubation of the gland in sucrose solution (Fig 8E and 8F). Moreover, after 40 min finishing the injection of NaFluo through the lateral brain ventricle, followed by the administration of Evans blue dye through the left heart ventricle, it was observed an accumulation of NaFluo in the PN and PI, excluding PD. This result suggests an infiltration and accumulation of the fluorescent dye dissolved in the CSF, and the Evans blue was observed at the periphery of the whole pituitary (Fig 8G and 8H).
Regarding the injection of the 2-μm fluorescent beads, these were observed at the PN edge and in the Rathke´s cleft (Fig 9Aa) and at the PD border (Fig 9Ab). When 0.2-μm fluorescent beads were injected, they accumulated at the posterolateral side of the PD (Fig 9Ac), but they were seen only on the periphery as observed in the 3D image video (S3 Fig). Within the Rathke´s cleft there are cells suspended in colloid (Fig 9Ad and 9Ae). When 2-and 0.2 μm fluorescent beads were injected intracerebroventricular, they entered the cleft and were phagocytosed by cells inside the cleft space (Fig 9Ac, and S4 Fig). After the administration of NaFluo through the left lateral brain ventricle and at different time intervals, various intracranial tissues were obtained, and their NaFluo accumulation was measured: pituitary, media eminence (EM), cerebellum cortex (CER) and optical nerve (ON). From the pituitary, only the PD was analysed. It is important to emphasize that the samples were washed three times with PBS after they were processed. The initial fluorescence level was the endogenous fluorescence. Following intracerebroventricular NaFluo administration, a statistically significant increase in fluorescence emission in all the samples was observed from 10 min after the infusion until 30 min later (Fig 9B). This increment was 113 and 63 times more in the PD, 319 and 512 times more in the EM, 65 and 98 times more in the CER, and 45 and 154.5 times more in the ON at 10 and 30 min, respectively. The accumulation of fluorescein after 10 min of NaFluo administration was easily observable macroscopically and showed clear differences between the samples studied (S5 Fig).

Discussion
Our results showed that the rat hypophysis capsule has different thicknesses around the gland. At the posterior-coronal side, the capsule is structured by a membrane with different layers of fibroblast-like cells, a loose mesenchymal membrane with cells with large pale nucleus, followed by an intricate layer of membrane processes rich in capillary vessels and at the more external surface a fibrous layer, suggesting that at this level the capsule corresponds to a leptomeninges organization [17,18]. Furthermore, Krish and Buchheim [4] described the rat capsule as a meningeal membrane. However, unlike brain leptomeninges in mice [17], the pituitary has an external fibrous layer and fibroblast-like cells densely packed in a fibrous membrane. The human pituitary envelope has been studied more extensively [19], and it has been described to consist of two membrane layers: a lamina propria covering the external surface of the gland, and an outer fibrous layer, the capsule, with a space between them [3]. Moreover, the capsule is thicker at the inferolateral region than the other parts of the gland [3]. SEM showed that the capsule is thick at the PN and the PI but is not a continuous layer because the membrane passes through a space between both lobes. A micrograph obtained by Wada et al. [20] shows a complex organization of the capsule at this level. However, the capsule at the PI continues towards the PD, and although it is less thick, it is structured by different layers of fibroblast-like cells, and a dense fibrous layer with cell characteristics similarly to those present at the thick capsule of the PI. Then, the gross capsule that resembles the leptomeninges also covers part of the PD; however, at the more anterior-rostral side of the gland, there seems to be a thin layer of fibroblast-like cells immersed in a thin fibrous membrane. According to Mesey and Miklós [1], only PN and PT are covered by leptomeninges, but Guerra et al. [8] showed Differences in fluorescence between the tissue were analyzed by one-way analysis of variance (ANOVA) followed by that at PT the arachnoid is loose, leaving only the pia mater membrane covering this part of the pituitary. Our observations suggest that the pia mater continues to the PD, in agreement with the observations of Guerra et al. [8] in the PT and the conclusion of Ciric [2] in human pituitaries. The relationship between the capsule and the pituitary parenchyma shows that they are separated by a basal membrane or lamina propria covering the pituitary surface, as can be observed in this study and what was reported by Krish and Buchheim [4]. Moreover, we observed that the membrane covering the surface is attached to the surface of the superficial tissue, as was reported by Krisch and Buchheim [4]. Nevertheless, Schechter [21] observed that there are discrete zones at the parenchymal border where gonadotrophs protrude through the basal membrane and could release factors to the meningeal capsule, such as fibroblast growth factor. The basal membrane and the capsule are separated, and CSF fills the space, in accordance with our observations with the presence of Evans blue and the polystyrene pearls at the borders of the gland, and with the measures of NaFluo accumulation at the surface of the PD. We also observed the presence of cells where a cilium protrudes towards this space, suggesting that they can receive signals from the CSF. According to the volume transmission communication concept, the pituitary could receive messages from different brain areas through the CSF and, as Veening and Barendregt [12] postulate, it could respond in accordance with the different brain states. Jindatip et al. [22] described a novel pituitary cell that is desmin-positive and exhibits a cilium; however, they reported that this cell is localized in the capillary vicinity. Not only is the surface of the pituitary bathed with CSF, but we could also localize the Evans blue dye and polystyrene beads inside the pituitary cleft and NaFluo accumulated in PN and PI after they were infused through the lateral ventricle of the brain. It was interesting to find the presence of the 2-μm diameter fluorescent pearls in this space. The entrance of the CSF to the pituitary cleft could be explained if it is considered a continuation of the PT cisterns to the cleft [8,23]. Another characteristic present in the overall capsule is the presence of capillaries, which are abundant and correspond to the capsular network [24]. According to Murakami et al. [24], the capillary capsular network receives its arterial twigs mainly from the internal carotid and posterior hypophyseal arteries and is connected into the neurohypophyseal, dorsal adenohypophyseal and ventral adenohypophyseal veins. According to our observations when Evans blue was administered intracardially, this capillary network is vessels that are continuous non-fenestrated, as Krish and Buchheim [4] reported and we also observed. Considering that these vessels are in a meningeal context, it is interesting to note that they are capillaries, differing from the rest of the pial vasculature which are arterioles and venules [25], suggesting that they can regulate the free movement of molecules. Further studies are necessary to characterize the function of these capillaries.
When we looked for the cell organization of the epithelia covering the pituitary cleft, also named the Marginal Cell Layer (MCL), and in accordance with Ciocca [10] and Correr and Mota [11], we observed an abundance of ciliated cells at the posterior face localized forward to PI, and the PD-side exhibits an epithelium with fewer multiciliate cells. However, at both epithelial layers there are other cells with multiple microvilli and others presenting a cilium at their apical surface. It was interesting to find that cells below the epithelia are clustered with the multiciliate and microvilli cells forming a unit. In accordance with Ishii and Ishibashi [26], we observed that no adhesion complexes are present between the epithelial and the cells below them. Moreover, they are separated from endocrine cells by a connective thin membrane. These units are more defined at the PI-side epithelial lining of the Rathke´s cleft, but at the Holm-Sidak´s multiple comparisons test. Differences between endogenous and NaFluo accumulation MR and AR groups, * P <0.05, ** P <0.01, ** P <0.0001.
https://doi.org/10.1371/journal.pone.0286399.g009 PD-side, these units are rich in capillaries. The observations at the TEM showed epithelial cells at the surface jointed with the typical epithelial adherence complexes: zonulae occlude, zonulae adhere and desmosomes, and the cells below them have abundant mitochondria. Together with this epithelium are cells that present a cilium, as seen in the rest of the superficial layer of the PD. The data suggest that the epithelia lining the surface of the cleft moves an external liquid medium in addition to sensing its composition. Supporting this suggestion is our observation of dyes dissolved in the CSF inside the pituitary cleft. Moreover, the epithelia of both layers of the cleft express aquaporin 4, and specifically, the multiciliate cells express aquaporin 5 [27]. Moreover, it has been reported that multiciliate epithelial cells of the MCL express the transcription factor FOXJ1, an essential expression factor for ciliogenesis, and together with the transcription factor SOX2 maintains the phenotype of these cells [28]. The expression of aquaporin 4 in the brain is well known to be involved in fluid transport into and out of it; therefore, it is an important participant in the volume control of the brain [29]. However, a recent study by Horiguchi et al. [30] showed another function of ciliated cells: they express the enzymes that synthesize and degrade retinoic acid and they suggested that as they adjust the retinoic acid concentration, they regulate the stem cell niche of the pituitary cleft. At the border of the pituitary cleft, constituted by the MCL, it is well known that there are SOX2+ progenitor cells [31].
It has been established that the principal regulator of the pituitary is the hypothalamus, which releases factors to the portal vessels, where they reach the pituitary cells. These factors are messengers of the integratory response of the nervous system and control the secretory function of the pituitary cells. Other inputs for the pituitary arrived by circulating blood. The efficient response of the pituitary is ensured by its tissue organization in homotypic and heterotypic networks [32,33]. In the PT, Guerra et al [8] observed that the CSF bathes its interior by a cistern system. This observation is important because PT is recognized as a structure for the regulation of seasonal rhythms for reproduction and is related to metabolic and immune functions, which respond to melatonin and its photoperiod and temperature variations through thyroid stimulating hormone (TSH) [34,35]. Melatonin reaches its target tissues in two ways, the bloodstream and CSF, and through the latter, a strong stimulus is achieved [36]. Our observations show CSF bathing the pituitary cleft, suggesting that this communication path could be used by the PI and PD with the PT and median eminence system. Moreover, Horiguchi et al. [30] showed that epithelial cells of the posterior layer synthesize retinoic acid, and melatonin is known to stimulate its synthesis in tanycytes participating in the circannual regulation of body weight and breeding [37]. Moreover, we hypothesized that signals from the CNS can reach the secretory parenchymal cells of the PD travelling in the CSF and be recognized by the ciliated cells present on the surface of the PD and the epithelium of the pituitary cleft. Recognized pituitary cells exhibiting a single cilium are localized: at the PT and are secretory cells [8], at the pituitary cleft [10], at the PD in the parenchymal follicles and are folliclestellate cells [38], and at the blood vessel periphery and are desmin-immunopositive cells [22]. We now added a non-identified cell at the surface of the PD and facing the pituitary capsule. What is the physiological function of these peripheral cells and how do they transmit what they perceive? According to the volume transmission communication, through the CSF neuroactive substances travel nearby or long distances, reaching the ventricular epithelia and outer surface of the brain and inducing the secretion of neuromodulators which contribute to regulate brain states [12,13]. The cellular function of the ciliate peripheral cells and those of the Rathke´s cleft epithelia we have described could be the transmission pathway of CSF signals to the pituitary coordinating it with other brain areas. However, further studies must be performed. Moreover, it is recognized at the PD an intricate network of follicle-stellate cells, which are characterized as a heterogeneous population with different electrical responses and present gap junctions allowing a large-scale intrapituitary communication [39]. Interestingly, Sato et al. [40] described that follicle-stellate cells are abundant at the basal periphery zone and at the cranial and caudal regions. According to our observations, at the caudal region, the capsule is thick and rich in capillaries, suggesting a zone of interchange between blood and CSF. Our study showed that the CSF bathes the pituitary gland suggesting that there is another way to intercommunicate the CNS and the gland through the CSF.